Consumption of excess energy, high fat diets are a useful model for investigating the mechanisms that lead to obesity and Type 2 Diabetes. Ceramides have been shown to interfere with insulin signalling and are elevated in skeletal muscle of obese, insulin resistant individuals (Adams et al., 2004). We aimed to investigate if the expression of skeletal muscle ceramide species was altered in healthy males following consumption of a diet designed to lead to insulin resistance. Twenty healthy males (22 ± 1 y; 71.24 ± 2.16 kg (mean ± SEM) were matched to one of two groups. Both groups participated in 6d high fat overfeeding (150% of total kcal, 60% FAT, 25% CHO, 15% PRO) but one group received 10% of their fats from fish oil sources (FO) while the other consumed mostly saturated and monounsaturated fats (HF). The overfeeding period was bookended by two identical trial days. Following an overnight fast, participants provided a basal blood sample and skeletal muscle biopsy. The blood sample was used to measure glucose and insulin concentrations for calculation of insulin sensitivity indices (Matsuda Index and HOMA-IR) while the skeletal muscle biopsy was used to assess the expression of ceramide species by LCMS-MS (results expressed as nmol/mg wet muscle). Statistical analyses were conducted using a repeated measures 2-way ANOVA with statistical significance set at p < 0.05. As no significant differences were observed between groups, all data presented herein refer to n = 20 (mean ± SEM) and p values are related to changes in response to the overfeeding period. Total ceramide expression was greater following the overfeeding period (8.52 ± 1.24 to 12.09 ± 1.73, p = 0.02) with significant increases in 5 of 11 species (C16:0: 0.39 ± 0.05 to 0.47 ± 0.06, p = 0.049; C22:0: 1.20 ± 0.23 to 1.82 ± 0.35, p = 0.02; C23:0: 0.56 ± 0.12 to 0.86 ± 0.17, p = 0.01; C24:1: 1.88 ± 0.38 to 3.19 ± 0.55, p = 0.02; C24:0: 1.98 ± 0.33 to 2.71 ± 0.46, p = 0.03). However, irrespective of group, 6d high fat overfeeding failed to induce any decrements in whole body insulin sensitivity (Matsuda: 3.45 ± 0.27 to 3.24 ± 0.24, p = 0.61; HOMA-IR: 3.13 ± 0.31 to 2.67 ± 0.60, p = 0.40). High fat overfeeding is associated with significant increases in total ceramide expression within skeletal muscle without any changes in whole body insulin sensitivity. Given the association between ceramide expression and impaired insulin signalling our data potentially offer insight into early adaptations that occur in the period preceding the development of insulin resistance.